The endothelial lining of the brain microvasculature, also known as the blood-brain barrier (BBB), constitutes a physical and metabolic barrier that tightly regulates brain uptake of ions, small molecules, proteins, and circulating cells. Since brain endothelial cell plasma membranes contact both the bloodstream and brain interstitial fluid, they are ideally positioned to act as the controlling interfaces for signaling, immune regulation, and transport between the blood and brain. Therefore, many of the unique characteristics of the BBB endothelium are likely attributable to the protein composition of its plasma membranes. Moreover, the glycosylation status of plasma membrane proteins can significantly affect function and can be differentially regulated in disease. Plasma membrane protein profiling is necessary to identify these phenotypic determinants and assess their responses in neurological disease conditions. Unfortunately, technological limitations have restricted the coverage of the BBB plasma membrane proteome and glycoproteome, and specific binding reagents that can facilitate functional analysis are lacking. Thus, in this proposal we describe a new strategy that employs lamprey VLR antibodies to profile the BBB plasma membrane proteome and glycoproteome. Lampreys and humans last shared a common ancestor >500 million years ago, and due to this tremendous evolutionary divergence, even highly conserved mammalian proteins and carbohydrates are immunogenic in lampreys. By leveraging these unique aspects of the lamprey immune system, we anticipate that the proposed research will provide new insights regarding the BBB plasma membrane proteome and glycoproteome while simultaneously supplying highly specific lamprey VLR antibodies that can be used to functionally characterize BBB components. To achieve these goals, freshly isolated mouse brain microvessel plasma membrane preparations have been used to immunize lampreys, and the resultant VLR antiserum clearly recognizes the in vivo BBB and possesses a unique glycan-binding signature. This immune VLR repertoire has been extracted from the lymphocyte cDNA of immunized lampreys and used to construct a yeast display library. The resultant yeast display VLR library will be rapidly mined for monoclonal, BBB-binding VLR antibodies by flow cytometry. In parallel, an innovative glycochip panning technology will be used to screen for monoclonal VLR binders to BBB glycoforms. Finally, the cognate BBB antigens recognized by the resultant monoclonal VLR antibodies will be identified by mass spectrometry analysis of the immunoprecipitated antigen and by glycan microarray analysis. Such advancements in elucidating the BBB plasma membrane proteome and glycoproteome in combination with the generation of VLR targeting reagents may enable future translational applications such as targeting of BBB plasma membrane proteins for therapeutic drug delivery purposes or blockade of BBB components that promote neuroinflammatory diseases.